Throughout this specification, various patents, published applications and scientific references are cited to describe the state and content of the art. Those disclosures, in their entireties, are hereby incorporated into the present specification by reference.
The present invention is directed to a novel protein structures, termed a “polyvalent protein complex” or PPC, that comprise three or four antigen binding sites (ABS). These PPC comprise novel properties, such as trivalence and tetravalence, when compared to immunoglobulins and can substitute for immunoglobulins or other engineered antibodies in applications such as diagnosis, detection, and therapy of normal (ectopic) or diseased tissues. These diseased tissues include cancers, infections, autoimmune diseases, cardiovascular diseases, and neurological diseases. Normal tissues can be detected and/or ablated, such as when they are ectopic (misplaced, such as parathyroid, thymus, endometrium) or if they need to be ablated as a therapy measure (e.g., bone marrow ablation in cancer therapies).
Discrete VH and VL domains of antibodies produced by recombinant DNA technology may pair with each other to form a heterodimer (recombinant Fv fragment) with binding capability (U.S. Pat. No. 4,642,334). However, such non-covalently associated molecules are not sufficiently stable under physiological conditions to have any practical use. Cognate VH and VL domains can be joined with a peptide linker of appropriate composition and length (usually consisting of more than 12 amino acid residues) to form a single-chain Fv (scFv) with binding activity. Methods of manufacturing scFvs are disclosed in U.S. Pat. No. 4,946,778 and U.S. Pat. No. 5,132,405. Reduction of the peptide linker length to less than 12 amino acid residues prevents pairing of VH and VL domains on the same chain and forces pairing of VH and VL domains with complementary domains on other chains, resulting in the formation of functional multimers. Polypeptide chains of VH and VL domains joined with linkers between 3 and 12 amino acid residues form predominantly dimers (termed diabodies). With linkers between 0 and 2 amino acid residues, trimers (termed triabody) and tetramers (termed tetrabody) are in favor, but the exact patterns of oligomerization appear to depend on the composition as well as the orientation of V-domains (VH-linker-VL or VL-linker-VH), in addition to the linker length. Monospecific diabodies, triabodies, and tetrabodies with multiple valencies have been obtained using peptide linkers consisting of 5 amino acid residues or less. Bispecific diabodies, which are heterodimers of two different polypeptides, each polypeptide consisting of the VH domain from one antibody connected by a short peptide linker to the VL domain of another antibody, have also been made using a dicistronic expression vector that contains in one cistron a recombinant gene construct comprising VH1-linker-VL2 and in the other cistron a second recombinant gene construct comprising VH2-linker-VL1. (Holliger et al., Proc. Natl. Acad. Sci. USA (1993) 90: 6444-6448; Atwell et al., Molecular Immunology (1996) 33: 1301-1302; Holliger et al., Nature Biotechnology (1997) 15: 632-631; Helfrich et al., Int. J. Cancer (1998) 76: 232-239; Kipriyanov et al., Int. J. Cancer (1998) 77: 763-772; Holiger et al., Cancer Research (1999) 59: 2909-2916]. More recently, a tetravalent tandem diabody (termed tandab) with dual specificity has also been reported (Cochlovius et al., Cancer Research (2000) 60: 43364341]. The bispecific tandab is a dimer of two homologous polypeptides, each containing four variable domains of two different antibodies (VH1, VL1, VH2, VL2) linked in an orientation to facilitate the formation of two potential binding sites for each of the two different specificities upon self-association.
Methods of manufacturing monospecific diabodies, monospecific triabodies, monospecific tetrabodies and bispecific diabodies by varying the length of the peptide linker as described above are disclosed in U.S. Pat. No. 5,844,094, U.S. Pat. No. 5,837,242, and WO 98/44001.
Alternative methods of manufacturing multispecific and multivalent antigen-binding proteins from VH and VL domains are disclosed in U.S. Pat. No. 5,989,830 and U.S. Pat. No. 6,239,259. Such multivalent and multispecific antigen-binding proteins are obtained by expressing a dicistronic vector which encodes two polypeptide chains, with one polypeptide chain consisting of two or more VH domains (from the same or different antibodies) connected in series by a peptide linker and the other polypeptide chain consisting of complementary VL domains connected in series by a peptide linker.
Increasing the valency of a binding protein is of interest as it enhances the functional affinity of that protein due to the avidity effect. The increased affinity enables the resulting protein to bind more strongly to target cells. Furthermore, the multivalency may, via crosslinking, induce growth inhibition of target cells (Ghetie, et al, Blood, 97: 1392-8, 2001) or facilitate internalization (Yarden, Proc. Natl. Acad. Sci., USA, 94: 9637, 1990), either property is desirable for an anti-tumor agent. The present invention addresses the continuous need to develop multivalent, multispecific agents for use in therapeutic and diagnostic applications.
Another area of the present invention is in the field of bio-assays. Virtually every area of biomedical sciences is in need of a system to assay chemical and biochemical reactions and determine the presence and quantity of particular analytes. This need ranges from the basic science research lab, where biochemical pathways are being mapped out and their functions correlated to disease processes, to clinical diagnostics, where patients are routinely monitored for levels of clinically relevant analytes. Other areas include pharmaceutical research, military applications, veterinary, food, and environmental applications. In all of these cases, the presence and quantity of a specific analyte or group of analytes, needs to be determined.
For analysis in the fields of chemistry, biochemistry, biotechnology, molecular biology and numerous others, it is often useful to detect the presence of one or more molecular structures and measure binding between structures. The molecular structures of interest typically include, but are not limited to, cells, antibodies, antigens, metabolites, proteins, drugs, small molecules, proteins, enzymes, nucleic acids, and other ligands and analytes. In medicine, for example, it is very useful to determine the existence of a cellular constituents such as receptors or cytokines, or antibodies and antigens which serve as markers for various disease processes, which exists naturally in physiological fluids or which has been introduced into the system. Additionally, DNA and RNA analysis is very useful in diagnostics, genetic testing and research, agriculture, and pharmaceutical development. Because of the rapidly advancing state of molecular cell biology and understanding of normal and diseased systems, there exists an increasing need for methods of detection, which do not require labels such as fluorophores or radioisotopes, are quantitative and qualitative, specific to the molecule of interest, highly sensitive and relatively simple to implement.
Numerous methodologies have been developed over the years to meet the demands of these fields, such as Enzyme-Linked Immunosorbent Assays (ELISA), Radio-Immunoassays (RIA), numerous fluorescence assays, mass spectroscopy, colorimetric assays, gel electrophoresis, as well as a host of more specialized assays. Most of these assay techniques require specialized preparations, especially attaching a label or greatly purifying and amplifying the sample to be tested. To detect a binding event between a ligand and an antiligand, a detectable signal is required which relates to the existence or extension of binding. Usually the signal is provided by a label that is conjugated to either the ligand or antiligand of interest. Physical or chemical effects which produce detectable signals, and for which suitable labels exist, include radioactivity, fluorescence, chemiluminescence, phosphorescence and enzymatic activity to name a few. The label can then be detected by spectrophotometric, radiometric, or optical tracking methods.